A continuing research effort is being maintained in and attempt to understand the role played by immunoglobulin antibodies in the mechanism of the antigen-antibody immunological reaction. The antigen-antibody reaction serves as a defense against the effects produced when a host body, human or animal, is invaded by microorganisms or other foreign bodies which cause infections, diseases or toxic reactions. This reaction is also relied upon to detect the presence or absence of either antigens or antibodies in human or animal sera. The presence of antigen molecules, such as honeybee venom, in a host body induces the production of an antibody called immunoglobulin. The antibody molecule has reactive sites which are capable of reacting with reactive sites on the antigen molecule to produce an immunological complex. The critical factor in the antigen-antibody reaction is that the antibody will combine specifically with the induced antigen.
The three major classes of immunoglobulin antibodies in human serum are immunoglobulin G (IgG), immunoglobulin A (IgA) and immunoglobulin M (IgM). Two other classes of distinct immunoglobulins are immunoglobulin E (IgE) and immunoglobulin D (IgD). IgG is the most abundant antibody in human serum and consists of two pairs of polypeptide chains linked by disulfide bonds. The peptide chains are oriented in the shape of the letter "Y". The reactive sites on the molecule are represented by the ends of the two arms of the Y-oriented antibody.
IgA is the second most abundant immunoglobulin in human serum while IgM is the largest immunoglobulin and the first to appear during primary immune response. IgD and IgE are present in only minor amounts in normal human serum. IgE functions as a mediator of hypersensitivity such as the allergic reaction to honeybee venom (HBV). After binding to the HBV antigen, it causes the release of histamine and its attendant problems. Therefore, the determination of IgE plays an important part in the diagnosis of the atopic state and for measuring response to treatment. As a consequence, numerous methods have been suggested for quantitating serum IgE levels. Among some of the more important methods for determining IgE levels are radial immunodiffusion, double antibody precipitation, a variety of microtiter solid phase radioimmunoassays (MSPRIA) using various coupled bodies, and an enzyme-linked immunosorbent assay technique (ELISA).
In the (MSPRIA) technique for determining specific IgE, the assay is carried out in flexible polyvinyl chloride "u" microtiter test plates by sequentially incubating antigen, albumin, test sera, and finally radiolabeled rabbit or goat antihuman IgE. Wells are cut free of the test plate with a hot wire-cutting apparatus and counted individually in a gamma counter. The amount of radioactivity bound is then proportioned to the amount of specific IgE in the serum. The MSPRIA technique is antigen and antibody specific. Rye-specific IgE levels assayed with the MSPRIA technique correlated with quantitative end point titration skin tests using perennial rye antigen. The rye-specific IgE levels assayed by MSPRIA also correlated with those assayed by RAST with a correlation coefficient of 0.95. The MSPRIA is well suited for mass screening and represents a useful method for measuring specific IgE. The (ELISA) assay technique for determining specific IgG is similar to the (MSPRIA) technique but with some differences. In this technique the assay is performed in polyvinyl chloride "u" microtiter plates having a series of test wells in which an antigen is incubated and absorbed on the walls of the polyvinyl chloride test wells. The antigen is sequentially overlaid with human albumin serum (HSA), unknown test serum diluted in 10% normal goat serum (NGS) and peroxidase-labeled goat or rabbit anti-human IgG in (NGS). O-Phenylenediamine (OPD) was used as a substrate. After incubation, the color reaction from the (OPD) was stopped and read on a spectrophotometer. A quantitation of the specific IgG was made by comparison to a known reference curve having arbitrarily assigned quantities of the specific IgG.
The (MSPRIA) and (ELISA) techniques have proved successful since the antigens or antibodies can absorb to a microtiter polyvinyl plate without the need of chemical modification for bonding. In addition, the plastic plates are of convenient size for handling and washing procedures. Unfortunately, most of these tests have not been able to measure IgE quantitatively according to a known reference and are expressed semi-quantitatively. There is also considerable interference from IgG when making a determination for IgE. This makes test results less reliable and it makes it difficult to compare one assay to another.
With the present invention, however, it has been found that modifying the (MSPRIA) or (ELISA) assay technique, in accordance with the techniques of this invention, reduces interference from specific IgG, a factor of particular importance to patients on immunotherapy; and is quantitative and referenced to a known and available standard (the NIH IgE reference). The modifications of this invention over that of the conventional (MSPRIA) AND (ELISA) techniques comprise the use of a monoclonal mouse anti-human IgE antibody which is labeled by using radioactive .sup.125 I in the case of the (MSPRIA) technique; and with peroxidase enzyme in the case of the (ELSIA) technique. Another basic difference resides in the fact that the supernatant fluid in the test well is serially transferred to additional test wells a sufficient number of times to materially and significantly reduce interference from the effects of IgG.
The modifications of the conventional (MSPRIA) and (ELISA) assay techniques as conceived by this invention, that is the serial transfer of supernatant fluid and the use of monoclonal mouse antihuman IgE (rather than affinity-column purified anti-IgE) are novel and provide the means for allowing very precise measurements and sensitivity of undiluted or low dilutions of serum samples.